1. Field of the Invention
This invention relates to a method for determining the level of expression of a target protein in a tissue or cell sample. Specifically, the invention relates to a method for determining the level of expression of a target protein in a tissue or cell sample using image analysis.
2. Background of the Invention
In aiding a clinician in the diagnosis of cancer, a pathologist faces two key problems. First, the pathologist must determine whether a tissue or cell sample removed from a patient is benign or malignant. Second, upon reaching a determination that the tissue or cell sample is malignant, the pathologist must then classify the aggressiveness of the cancer and determine its clinical and biological behavior.
A diagnosis of cancer must be confirmed through histological examination of a tissue or cell sample removed from a patient. Such histological examination entails tissue-staining procedures that allow the morphological features of the tissue to be readily examined under a light microscope. The pathologist, after having examined the stained tissue or cell sample, makes qualitative determinations of the state of the tissue or the patient from whom the sample was removed and whether the tissue is benign or malignant. The aggressiveness of the tumor, however, is difficult to ascertain using standard histological techniques. The clinician uses the pathologist's histological analysis to select a suitable treatment, balancing the resistance or responsiveness of the cancer to therapy with the potential harm to the patient resulting from the selected therapy (Muss et al., 1994, N. Engl. J. Med. 330:1260-66).
Visual examination of tissue and cell samples is often augmented by the use of an automated (computer-aided) image analysis system. A representative system includes a computer that receives a magnified image of the tissue or cell sample from a television camera and processes the received optical image. Image analysis is generally used to assess the affinity of stains for various biological markers. Examples of suitable affinity stains include chromagen-labeled monoclonal antibodies directed against the estrogen receptor (ER), the progesterone receptor (PR), the HER-2/neu protein, and the epidermal growth factor receptor (EGFR).
The coupling of affinity staining and computer-aided image analysis has permitted clinicians to better select optimal therapies for their patients (e.g., hormone therapy for cancers that are ER and PR positive and anti-oncogene receptor therapy—such as using monoclonal antibodies directed against to HER-2/neu (Herceptin™), EGFR, or C225™, alone or in combination with chemotherapy). In addition, image analysis techniques can be used to quantitate other receptors such as those in the erbB receptor family (HER-1, HER-2/neu, HER-3, and HER-4), their ligands (EGF, NDF, and TGFα), and downstream signals (PI3 kinase, Akt, MAP kinase, and JUN kinase) (National Institute of Health Consensus Development Conference: Steroid Receptors in Breast Cancer, 1979, Vol. 2 No. 6; Kraus et al., 1989, Proc. Natl. Acad. Sci. U.S.A. 86:9193-97; Mendelsohn, 1990, Semin. Cancer Biol. 1:339-44; Hancock et al., 1991, Cancer Res. 51:4575-80; Peles et al., 1991, EMBO J. 10:2077-86; Peles et al., 1992, Cell 69:205-16; Arteaga et al., 1994, Cancer Res., 54:3758-65; Pietras et al., 1994, Oncogene 9:1829-38; Baselga et al., 1999, Proceedings of AACR NCI EORTC International Conference, Abstract 98; Cobleigh et al., 1999, J. Clin. Oncol. 17:2639-48; DiGiovanna, 1999, PPO Updates: Princ. Practice Oncol. 13:1-9; Shak, 1999, Semin. Oncol. 26:71-77; Sliwkowski et al., 1999, Semin. Oncol. 26:60-70; Vincent et al., 2000, Cancer Chemother. Pharmacol. 45: 231-38).
When a tumor sample is examined using immunohistochemical analysis alone, accurate quantitation of a specific target protein being expressed by the tumor cells is often hampered by the presence of normal tissue that is removed from the patient with the tumor sample. Biological approaches for the treatment of cancer, such as the use of monoclonal antibodies or kinase inhibitors, rely on accurate quantitation of specific target proteins in a tumor sample. For example, the effectiveness of the HER-2/neu antibody Herceptin™ has been shown to depend on the amount of HER-2/neu being expressed in tumor cells (Table 1).
There remains a need in the art to develop more accurate methods for determining the level of expression of a target protein in a tissue or cell sample. The development of such methods would have wide application in the treatment of cancer.